Enterobacteria such as Escherichia coli and Salmonella enterica are facultative aerobes, and when cultured in the absence of oxygen can use a variety of alternative respiratory oxidants. Nitrate and nitrite are the preferred alternatives, and anaerobic respiratory enzyme gene expression is tightly controlled in response to their availability. This control is mediated by dual interacting two-component regulatory systems. Nitrate and nitrite control the autophosphorylation of two cytoplasmic membrane-bound sensor-kinases, the NarX and NarQ proteins, which in turn control the phosphorylation of two DNA-binding response regulators, the NarL and NarP proteins. Thus, transcription initiation at more than one dozen operons is activated or repressed according to electron acceptor availability. The Nar regulatory network is unique to enterobacteria; other species of proteobacteria (including several human pathogens) contain only the NarQ-NarP system (e.g., Vibrio cholerae) or the NarX-NarL system (e.g., Pseudomonas aeruginosa). The proposed studies therefore broadly inform our understanding of anaerobic physiology for many species within the gamma and beta subdivisions of the proteobacteria. Work proposed here comprises five specific aims: (1) We must characterize cross-regulation in vitro in biochemical detail in order to evaluate and extend models based primarily on in vivo observations. (2) In collaboration with E. P. Baldwin, we will study cooperative DMA binding by the response regulator NarL. (3) In collaboration with P. J. Kiley, we will study transcription activation by the response regulator NarP. (4) We continue to explore response to defined regulatory signals by the sensors NarX and NarQ. (5) In collaboration with M. M. Igo, we will identify previously- unknown target genes whose expression is controlled by the NarX-NarL or NarQ-NarP systems. Our overall goal is to integrate both in vivo and in vitro approaches to better understand the physiology of anaerobic metabolism. The relevance to public health of this fundamental research is in the realm of anaerobic physiology and metabolism. Most pathogenic species within the proteobacteria are facultative aerobes or facultative anaerobes, and many infectious diseases involve colonization of anaerobic environments such as the mammalian intestine. Thus, results from the model species E. coli enhance understanding for a broad range of pathogens that significantly impact public health.